Microfluidic cartridge for molecular diagnosis, docking station using a microfluidic cartridge, and process for analyzing a biological sample

ABSTRACT

A microfluidic cartridge for detecting one nucleic acid of a sample, including a plurality of functional volumes split into functional areas and a fluidic network of microchannels. At least three functional areas are fluidly connected to a central distribution hub of fluids by one or more hub-connected microchannels, the central distribution hub being capable of pumping and injecting fluids from a first functional area to a second functional area by passing through the central distribution hub; and at least three valves of hub-connected microchannels are arranged so that the at least three valves are adapted to be actuated mechanically by a single external cam-driven actuator. A docking station using such a microfluidic cartridge and a process for analyzing a biological sample involving a docking station and a microfluidic cartridge are also described.

The invention relates to the field of microfluidic devices used to makemolecular or biological diagnostics.

The invention more particularly relates to a microfluidic cartridge foranalyzing at least one nucleic acid contained in a biological sample.

The invention also relates to a docking station designed to use andoperate such a microfluidic cartridge.

The invention finally relates to a method of analysis of a biologicalsample implementing such a microfluidic cartridge.

Microfluidic devices designed for the search and the analysis of atleast one nucleic acid or one nucleotide sequence contained in abiological sample incorporate various means in order to: prepare thebiological sample to extract nucleic acids from said sample; amplify theat least one target nucleic acid from the extracted nucleic acids usingstandard amplification techniques like, for example, Polymerase ChainReaction (also known as <<PCR>>); and detect, e.g. optically, andanalyze the target nucleic acids using known molecular recognitionmechanisms like, for example, hybridization.

Therefore, in order to perform the analysis of the at least one nucleicof a sample, said sample needs to be transferred sequentially indifferent functional areas of the micro microfluidic cartridge, eachfunctional area being dedicated to a specific operation on the sample.

The documents WO 2009/049268 and US 2012/115738 describe, for example, amicrofluidic device comprising a plurality of functional areas: an areaof sample preparation for extraction of nucleic acids, a range ofnucleic acid amplification, and a surface analysis and detection ofamplified nucleic acids. Said detection area is likely to be a biochip.

In those documents, the microfluidic cartridge features very complexstructure so as to be modular and allow an easy and rapidreconfiguration in order to suit various applications. In particular,many pumps with shared-valve structure are implemented within each ofthe functional areas of the microfluidic cartridge to transfer fluidsfrom one functional area to the other.

Therefore, the cartridge of documents WO 2009/049268 and US 2012/115738presents a large volume and the transfer of fluids cannot be operated ina simple manner, with a limited number of actuators.

The widespread use of these devices, especially in the context ofmolecular diagnostics in humans, for which the cartridge must bediscarded after each use, is limited by the complexity and high costsinherent in this technology. Furthermore, these devices, such as the onedescribed in US 2012/0034705, often consisting of a variety of manyelements for achieving the various stages of analysis, they areextremely fragile and difficult to handle.

It is therefore desired that a microfluidic cartridge is massproducible, inexpensive, and most preferably disposable. However,because such microfluidic devices integrate complex steps of molecularanalysis, it may be difficult to properly coordinate various tasks ofconventional microfluidic devices. It is therefore also desired that themicrofluidic cartridge be simple to operate and that many orsubstantially all of the fluid processing steps be automated directly onthe microfluidic cartridge.

For that purpose, the present invention proposes a microfluidiccartridge making it possible, on the one hand, to integrate, within thelatter, not only all the fluids required for its operation, but also thewhole of microfluidic circuits, microchannels and valves, the reactionchamber and the biochip, and, on the other hand, to make transfers andmovements of fluids in a simple manner, in a reduced volume and by meansof a compact external actuator.

More precisely, the present invention provides a microfluidic cartridgefor detecting at least one nucleic acid of a sample, said microfluidiccartridge comprising:

-   -   a plurality of functional volumes split into functional areas        such as at least, a sample preparation area, a nucleic acid        amplification area, a nucleic acid analysis area, a waste area,        and    -   a fluidic network of microchannels,

wherein:

-   -   at least three functional areas are fluidly connected to one        central distribution hub of fluids distribution by one or more        hub-connected microchannels, each of said hubconnected        microchannels having a hub end and an area end, said central        distribution hub being capable of pumping and injecting fluids        from a first functional area to a second functional area of said        at least three functional areas by passing through said central        distribution hub, said second functional area being identical or        different from said first functional area, and    -   at least three valves, each located on a hub-connected        microchannel, are arranged in said microfluidic cartridge so        that said at least three valves are adapted to be actuated        mechanically by a single external cam-driven actuator.

The microfluidic cartridge according to the invention has thus theadvantage, thanks to the use of the central distribution hub of fluids,to facilitate the transfers of fluid from a first functional area to asecond functional area. This makes it possible to use only one simplefluid displacement system (typically a pumping system) for most of thefluid movements of the microfluidic cartridge, for inducingdepressurization and pressurization in order to displace the fluid froma functional volume or area to another one and to reduce the volume ofthe microfluidic cartridge.

The microfluidic cartridge comprises less moving elements and its costis therefore reduced compared to prior-art cartridges.

Moreover, the system for actuation of the valves of the microchannelsconnected to the central hub may also be more compact and simpler thanthe system disclosed in US 2012/0034705, thanks to the arrangement ofthese valves in the microfluidic cartridge.

In one embodiment, the at least three functional areas that areconnected to the central distribution hub are the sample preparationarea and the waste area.

In one embodiment, the at least three functional areas comprise thenucleic acid analysis area, and/or the nucleic acid amplification area.

In another embodiment, the at least three functional areas comprise thesample preparation area, the nucleic acid analysis area, and the wastearea.

In another embodiment, the at least three functional areas comprise allfunctional areas of the microfluidic cartridge.

In one embodiment, the microfluidic cartridge further comprises at leasttwo valves that are actuated by linear actuators and are independent ofthe cam-driven actuator.

The microfluidic cartridge according to the present invention may beseen as a “lab-on-a-chip” that can perform the complete nucleic acidanalysis of a sample, from sample collection to the reading of theresult, typically performed in the diagnostics or microbiologylaboratory.

The detection of the presence in the sample, of a nucleic acid ormolecular marker whose sequence is specific to a gene of interest, isunderstood as a molecular diagnostics in this application.

The microfluidic cartridge integrates usually over a few squarecentimeters several specialized functional areas and volumes performingcomplex analysis conventionally made using several laboratory apparatus.The advantages are that theses operations can be automated whileconsuming low reagents volumes.

Besides, other advantageous and non-limiting characteristics of themicrofluidic cartridge according to the invention are described below.The said characteristics correspond to various embodiments of theinvention that can be taken alone or in combination.

The at least three valves are spatially arranged in the microfluidiccartridge so that they are adapted to be actuated simultaneously by thesingle external cam-driven actuator. In particular embodiments of theinvention, said at least three valves are linearly or circularlyarranged in the microfluidic cartridge.

Typically, the said at least three valves are valves of hub-connectedmicrochannels, connecting the sample preparation area, the nucleic acidanalysis area, and the waste area to the central hub.

Preferentially, the said valves are located close to, or at the area endof the said hub-connected microchannels, said area end being the one ofthe two ends of the hub-connected microchannel which is turned towardsthe corresponding functional area. On the opposite, the hub end is theone of the two ends of the hub-connected microchannel which is turnedtowards the central distribution hub of fluids.

In one embodiment of the invention, each hub microchannel comprises onevalve located, close to, or at their area end. Said valves arepreferentially spatially arranged in order to be simultaneously actuatedby the external cam-driven actuator as mentioned above.

At least two functional areas of the plurality of functional areas canalso be directly fluidly connected to each other by one or morearea-connecting microchannels, each of the said area-connectingmicrochannels having at least a valve that is preferentially actuated bya linear actuator independent of the cam-driven actuator.

For example, said two functional areas are the nucleic acidamplification area and the nucleic acid analysis area.

In another example, the said two functional areas are the nucleic acidanalysis area and the waste area.

Typically, a microfluidic cartridge according to the invention isdisposable and comprises:

-   -   a cartridge plate comprising:        -   a substrate having a first face and a second face, a            plurality of grooves flush with the first or second surface            and a plurality of through holes connecting said first and            second surfaces, and        -   a first film bonded on first face of said substrate of the            cartridge plate, said grooves flush with the first face            being sealed by said first film to form the hub-connected            microchannels, said first film being a first deformable            membrane adapted to be deformed by an external actuator,    -   a cartridge body in contact with the cartridge plate on the        second surface of said substrate, said cartridge body        comprising:        -   a lateral wall which extends from the second surface of said            substrate, and        -   a plurality of internal walls which defines a plurality of            functional volumes of said cartridge body, and        -   a cartridge cover adapted to close the different functional            volumes.

In one embodiment, the cartridge plate comprises a second film bonded onthe second face of the substrate of the cartridge plate, the pluralityof grooves flush with the second face being sealed by said second filmto form the area-connected microchannels.

Typically, the cartridge plate comprises at least one recessed cavityformed in the substrate and extending from the first face.

Typically also, the first film bonded on the first face of the substratecloses said at least one recessed cavity to form at least one reactivechamber for nucleic acid amplification.

In a preferred embodiment, a micro-array slide (or biochip) bonded onthe first face of the substrate closes said at least one recessed cavityto form at least one detection chamber for nucleic acid analysis.

In a preferred embodiment, the microfluidic cartridge comprises asemi-permeable membrane between the cartridge body and the cartridgecover adapted to let air pass through it while preventing liquids toleak out of the functional volumes.

Typically, the functional volumes of the cartridge body encompassseveral functional areas (e.g.: at least a sample preparation area, anucleic acid amplification area, a nucleic acid analysis area and awaste area). Said functional volumes are containers adapted to receivetubes, fluids such as sample, reagent products, or a purificationcolumn.

In one embodiment, the central hub of fluid distribution comprises a hubbody and a plunger seal adapted to slide in and out of the hub body topump from or inject fluids in the functional areas of said microfluidiccartridge through the hub-connected microchannels.

In another embodiment, the central hub of fluid distribution comprisesalso a syringe having a plunger to which the plunger seal is attached.

The microfluidic cartridge is adapted to be inserted into a dockingstation, within equipment designed to perform at least the followingfunctions: thermal control, control of fluid flow, valves actuation andoptical detection.

The present invention also proposes a docking station intended to useand operate a microfluidic cartridge such as mentioned above.

Therefore, the present invention provides a docking station adapted toreceive a microfluidic cartridge according to the invention, comprising:

-   -   a cam-driven actuator adapted to simultaneously actuate the at        least three valves of hub-connected microchannels,    -   means for optical excitation of the micro-array slide of said        cartridge, and    -   means for optical detection of an optical signal that is        representative of said nucleic acid in the sample analyzed by        the cartridge.

In one embodiment, the docking station also comprises actuation meansadapted to actuate linear/independently actuated valves of saidmicrofluidic cartridge.

In a particular embodiment, the cam-driven actuator is arotational-motion actuator.

In another particular embodiment, the cam-driven actuator is alinear-motion actuator.

Preferentially, the cam-driven actuator of the docking station isdesigned to open, among the valves of the microchannels connected to thecentral hub, at most only one of said valves.

In a preferred embodiment, the docking station according to theinvention comprises sliding means adapted to slide the syringe of thecentral hub in and out of the hub body to pump from or inject fluids inthe functional areas of said microfluidic cartridge through thehub-connected microchannels.

It is also an object of the present invention to provide an apparatusfor analyzing a biological sample comprising such docking station andmicrofluidic cartridge according to the present invention, for analyzingat least a nucleic acid of a sample.

Furthermore, the microfluidic cartridge according to the invention isparticularly adapted to be used in a process for analyzing a biologicalsample.

Therefore, it is another object of the present invention to propose aprocess for analyzing a biological sample, comprising the steps of:

a) providing said biological sample into at least one functional volumeof a sample preparation area of a microfluidic cartridge according tothe invention,

b) allowing said biological sample to get into contact with at least onereagent and/or one purification column present in another functionalvolume of the sample preparation area by actuating at least one valvecontrolling the flow of fluids of microchannels,

c) recovering the product resulting of step b to obtain an isolated DNAsample,

d) transferring the isolated DNA sample to at least one functionalvolume of the nucleic acid amplification area,

e) allowing said isolated DNA sample to get into contact with at least areagent for amplification and closing the valves of the functionalvolume of the amplification area,

f) performing DNA amplification,

g) recovering amplified DNA obtained at step f) and transferring it toanother functional volume of the hybridization area by actuating atleast one valve controlling the flow of fluids of microchannels,

h) allowing said amplified DNA to get into contact with at least onecompound capable of hybridizing with said DNA in the hybridizationchamber, and

i) obtaining a microarray image and automatically analyzing it.

An embodiment of the invention will now be described in detail withreference to the drawings, in which:

FIG. 1 is a perspective view of a microfluidic cartridge in a preferredembodiment of the invention;

FIG. 2 is an exploded view of the microfluidic cartridge of FIG. 1,making appear the cartridge plate, the cartridge body and the cartridgecover;

FIG. 3 is a schematic view of a preparation tube for a biological sampleto analyze with the microfluidic cartridge of FIG. 1;

FIG. 4 is an exploded schematic view of a syringe that can be used inthe microfluidic cartridge of FIG. 1 to pump and inject the fluids;

FIG. 5 is a schematic view of an <<amplification mix>> tube to beinserted in the microfluidic cartridge of FIG. 1;

FIG. 6 is a detailed view of the cartridge body of FIG. 2;

FIG. 7 is a detailed view of the cartridge plate of FIG. 2;

FIG. 8 is a bottom view of the cartridge plate of FIG. 7;

FIG. 8A is a sectional view of FIG. 8 according to the section planeA-A;

FIG. 8B is a sectional view of FIG. 8 according to the section planeB-B;

FIG. 9 is a detailed view of the area referenced I in FIG. 8;

FIG. 9A is a sectional view of FIG. 9 according to the section planeA-A;

FIG. 10 is a detailed view of the area referenced I in FIG. 8;

FIG. 10A is a sectional view of FIG. 10 according to the section lineB-B;

FIG. 11 is a top view of the cartridge plate of FIG. 7;

FIG. 12 is a schematic view of the mounting of the microfluidiccartridge of FIG. 1 in a docking station in a preferred embodiment(mechanical part only);

FIG. 13 is a schematic view of a rotational-motion cam-driven actuatorusing balls to actuate the valves of the microfluidic cartridge;

FIG. 14 is a schematic view of a perforated actuator plate allowing theholding in place of the balls of the cam-driven actuator of FIG. 13;

FIG. 15 is a schematic view of a circular plate having a detentoperating in the mechanism of the cam-driven actuator of FIG. 13;

FIG. 16 is a detailed view of another example of cartridge plate.

It has been shown in FIG. 1 and FIG. 2, respectively, an assembled viewand an exploded view of a microfluidic cartridge 1 according to apreferred embodiment of the invention, in which the microfluidiccartridge 1 is herein a disposable cartridge. It is meant by this thatthe microfluidic cartridge 1 is intended to be disposed of and placed ina container intended for receiving biological wastes.

As shown in FIGS. 1 and 2, the microfluidic cartridge 1 comprises threemain elements, i.e.: the cartridge plate 10, the cartridge body 20 andthe cartridge cover 30.

The microfluidic cartridge 1 also comprises a sample tube 40 containinga sample S, at least an amplification-mix tube 50 and a syringe 60.

These different elements of the microfluidic cartridge 1 will bedetailed hereinafter.

The cartridge plate 10 of the microfluidic cartridge 1 first comprises asubstrate 100 such as the one shown in detail in FIG. 7.

This substrate 100 has substantially the shape of a thin blade and has afirst face 101 and a second face 102. The second face 102 is the facethat is turned toward the cartridge body 20 when the cartridge isassembled (see FIGS. 1 and 2).

The cartridge plate 10 may advantageously be made by injection moldingof a thermoplastic polymer material such as the cyclic olefin copolymers(COC) or the cyclic olefin polymers (COP). The cartridge plate 10 ishere preferably made of polypropylene (PP). The COC and COP areamorphous and transparent materials based on cyclic olefins, whosebiocompatibility is excellent. These materials allow the making of asealed connection with a membrane and/or adhesive patches. They may inparticular by chosen in the group comprising polycarbonate,polyacrylamide, polyethylene, polymethyl-methacrylate (PMMA),polydimetyl-siloxane (PDMS), polyvinyl chloride (PVC).

Preferably, the dimensions of the substrate 100 of the cartridge plate10 are approximately, lengthwise and widthwise, comprised between 50 and150 mm long, preferentially between 85 and 125 mm and 25 and 75 mm wide,preferentially between 40 and 60 mm. The thickness of the substrate 100is preferentially comprised between approximately 1 and 5 mm,preferentially between 1 and 2 mm.

Generally, the microfluidic cartridge 1 includes a fluidic network ofmicrochannels in which various fluids circulate and which each compriseat least one valve for controlling the circulation of such fluids in thecorresponding microchannels.

It will now be described, for the particular embodiment of themicrofluidic cartridge 1 shown in FIGS. 1 and 2, where and how areformed these microchannels and the associated valves with reference toFIGS. 7 to 11 showing various views of the cartridge plate 10 and thesubstrate 100 thereof.

Therefore, as shown in particular in FIGS. 7 and 8, the cartridge plate10 first includes a plurality of through holes, which are herein as amatter of reference in a total of thirty-four and which are referenced:

-   -   H1 to H14,    -   H0 c to H5 c, and H9 c, H10 c, and    -   H7 a, H7 b, H11 a, H12 a, H15 a to H15 d, H16 a to H16 d.

All these through holes extend through the substrate 100, between thefirst face 101 and the second face 102, and preferably perpendicularlyto these two faces 101, 102 (see for example FIGS. 9A and 10A,respectively, for the through holes H4, H4C, and the through holes H8,H16 a).

These through holes, opening on each of the first and second faces 101,102, fluidically connect elements from either face to each other. It ismeant by this that a fluid can circulate in these through holes, in onedirection as in the other.

For a proper understanding, the distinction will be made, in thefollowing description, between three different types of through holes(see FIGS. 7 to 10A):

-   -   the through holes with recess: these are the through holes        referenced H1 to H14 (see FIGS. 8, 9 and 9A), flow through them        is actuated by valves    -   the central through holes: these are the through holes        referenced H0 c to H5 c, H9 c and H10 c, and    -   the simple through holes corresponding to the remaining through        holes and referenced H7 a, H7 b, H11 a, H12 a, H15 a to H15 d,        H16 a to H16 d.

The through holes with recess, referenced H1 to H14 in FIGS. 7 and 8,each have, at their end turned toward the first face 101 of thesubstrate 100, a recess, referenced R1 to R14, respectively, in FIGS. 7and 8, cylindrical in shape, made at the surface of the first face 101of the substrate 100. This is, for example, illustrated in FIGS. 9A and10A, which are partial sectional views of the substrate 100, where thethrough holes H4 (FIG. 9A) and H8 (FIG. 10A) are shown with theirrespective recess R4 and R8.

The recesses R1 to R14 have:

-   -   a diameter comprised between 1 mm and 10 mm, preferentially        between 2 mm and 8 mm, preferably of about 4 mm, and    -   a depth comprised between 0.02 mm and 0.4 mm, preferentially        between 0.05 mm and 0.15 mm, preferably of about 0.1 mm.

The central through holes, referenced H0 c, H1 c, H2 c, H3 c, H4 c, H5c, H9 c and H10 c (see for example FIG. 8), which are close to eachother are arranged herein in a circle. The interest of such anarrangement will be seen hereinafter.

Besides, the cartridge plate 10 includes a first plurality of sixteengrooves, referenced G1 to G16 in FIGS. 8 to 10A. These first grooves G1to G16 are made in the vicinity of the first face 101 of the substrate100, in such a manner to flush with this first face 101. This may beobserved, for example, in FIGS. 9A and 10A, in which the grooves G4(FIG. 9A) and G8, G16 are shown.

Advantageously, these grooves G1 to G16 are parallel to the first face101 of the substrate 100, having a depth generally comprised between0.01 mm and 0.5 mm, preferentially between 0.2 mm and 0.4 mm, preferablyof about 0.3 mm.

The width of these grooves G1 to G16 is herein equal to about 0.5 mm.

In the particular embodiment of the microfluidic cartridge 1 shown inFIG. 1, it is observed that these first grooves G1 to G16 extend (seeFIG. 8):

-   -   either between a central through hole H0 c, H1 c, H2 c, H3 c, H4        c, H5 c, H9 c, H10 c and a recess R1 to R10: this is the case of        the grooves G1 to G10 (see for example FIG. 9A for the groove G4        between the central hole H4 c and the through hole G4 with its        recess R4);    -   or between the central through hole H0 c and a simple through        hole H11 a, H12 a: this is the case of the grooves G11 and G12;    -   or between two simple through holes: this is the case of the        grooves G15 and G16 that extend between the through holes H15 a        and H15 c, and between the through holes H16 a and H16 c,        respectively. Regarding these particular grooves G15, G16, it is        also observed that they respectively comprise on their way a        simple through hole H15 b, H16 b.

The grooves G6, G7, G8, G11 and G12 share a common part that connectseach of these grooves G6, G7, G8, G11, and G12 to the central hole H0 c,and form this way a branched structure.

As shown in FIGS. 7 and 11, the cartridge plate 10 finally comprises asecond plurality of eight grooves G11 a, G12 a, G13 a, G14 a, G15 a, G15b, G16 a, G16 b. These second grooves G11 a, G12 a, G13 a, G14 a, G15 a,G15 b, G16 a, G16 b, are made in the vicinity of the second face 102 ofthe substrate 100, in such a manner to flush with this second face 102.This may be observed, for example, in FIG. 10A, in which the groove G16a is shown.

As for the first grooves G1 to G16, these second grooves G11 a, G12 a,G13 a, G14 a, G15 a, G15 b, G16 a, G16 b, are advantageously parallel tothe second face 102 of the substrate 100. They have the same dimensionalcharacteristics as the first grooves G1 to G16.

Generally, and as it can be understood by observing FIG. 8 (bottom viewof the substrate 100) and FIG. 11 (top view of the substrate), thesecond grooves G11 a, G12 a, G13 a, G14 a, G15 a, G15 b, G16 a, G16 b,formed on the second face 102 of the substrate 100 extend:

-   -   either between a through hole H6, H8, H11, H12, H13, H14, with        recess, and a simple through hole H15 a, H16 a, H11 a, H12 a,        H15 b, H16 b, respectively: this is the case, for example, of        the second grooves G11 a, G12 a, G13 a, G14 a, G15 a and G16 a        (see for example FIG. 10A);    -   or between two simple through holes H15 c, H15 d, H16 c, H16 d:        this is the case for example of the groove G15 b (between the        simple through holes H15 c, H15 d) and of the groove G16 b        (between the simple through holes H16 c, H16 d).

The through holes, recesses and grooves made in the above-mentionedsubstrate 100 are intended to form, on the one hand, the fluidic networkof microchannels, and on the other hand, the fluid control valves inthese microchannels.

For that purpose, it is understood that it is necessary to close thethrough holes, recesses and grooves that are, as just described, open tothe first surface 101 or the second surface 102 of the substrate.

Therefore, the cartridge plate 10 first comprises a first film 11 (seeFIG. 2) that, when the microfluidic cartridge 1 is assembled (see FIG.1), is located on the first face 101 of the substrate 100 of thecartridge plate 10.

Moreover, the form and dimensions of this first film 11 are adjusted soas (see FIG. 8):

-   -   to follow the outer profile 103 of the substrate 100, and    -   to extend over a large half of the substrate 100 so as to cover        the whole of the first grooves G1 to G16, the recesses R1 to        R14, and the central through holes H0 c to H5 c, H9 c, H10 c,        and the simple through holes H11 a, H12 a, H15 a to H15 c, and        H16 a to H16 c.

The first film 11 is preferentially made in a material similar to therigid substrate 100 of the cartridge plate 10. Generally, the first film11 is here made of polypropylene (PP).

Preferentially, the first film 11 is a thermoplastic film of about 0.1mm thick, bonded or welded to the surface of the first face 101 of thesubstrate, by thermo-welding, e.g. by laser-welding, bonding, adheringor chemical linking methods. This first film 11 closes the first face101 and provides the thickness of the microfluidic circuit.

Thus positioned and fixed on the first face 101 substrate 100, the firstfilm 11 closes and tightly seals the first grooves G1 to G16, therecesses R1 to R14, and the central through holes H0 c, H1 c a H5 c, H9c, H10 c, and the simple through holes H11 a, H12 a, H15 a to H15 c, andH16 a to H16 c.

In other words, the first film 11 cooperates with the first grooves, thethrough holes and the recesses to form a plurality of microfluidicschannels, or microchannels, and valves.

As shown in FIG. 12, microchannels C1 to C12, C15, C16, are thus formedby the closing of the first grooves G1 to G12, G15, G16, flush with thefirst face 101 of the substrate 100 by means of the first film 11deposited on this first face 101.

In the same manner, the valves V1 to V14 are formed by the deformablefirst film 11 placed opposite a valve seat formed by the recessed R1 toR14 formed at the surface of the first face 101 of the substrate 100.

In a preferred manner, the surface of the deformable first film 11,placed opposite the recesses R1 to R14 is, at rest, approximately planarand parallel to the first face 101 of the substrate, and capable ofbeing deformed by an external actuator (see infra). The deformation ofthe first film 11 at the level of the recesses R1 to R14 under theaction of this external actuator allows opening or closing the valves V1to V14.

More precisely, the deflection of the first film 11 opposite each valveseat, i.e. each recess R1 to R14, allows the obturation of thecorresponding through holes H1 to H14, whose diameter is far lower thanthat of each recess R1 to R14. This allows the making of a maximumobturation of the cartridge plate 10 while using a first film 11 havingcertain rigidity.

The cartridge plate 10 also comprises a second film 12 (see FIG. 2), ora plate, that, when the microfluidic cartridge 1 is assembled (see FIG.1), is located on the second face 102 of the substrate 100 of thecartridge plate 10.

The second film 12 is herein made of a material similar to the rigidsubstrate 100 of the cartridge plate 10 and its thickness, of about 0.1mm.

Alternatively, a plate may be used. This plate can have dimensionscomprised between 0.05 mm and 2 mm.

The second film 12 is bonded to the second face 102 of the substrate 100by bonding.

As a variant, the second film may be fixed on the second face bythermo-welding, adhering or chemical linking methods.

This second film 12 closes the second face 102 and allows the tightnessof the microfluidic circuit.

More precisely, the second plurality of grooves Gila, G12 a, G13 a, G14a, G15 a, G15 b, G16 a, G16 b, is closed and sealed by the second film12.

As for the first film 11, and as shown in FIG. 12, microchannels C11 a,C12 a, C13, C14, C15 a, C15 b, C16 a, C16 b, are thus formed by theclosing of the second grooves G11 a, G12 a, G13 a, G14 a, G15 a, G15 b,G16 a, G16 b, flush with the second face 102 of the substrate 100 bymeans of the second film 12 deposited on this second face 102.

The second film 12 has a rectangular opening 12A (see FIG. 2) so as toallow the passage thereof through the cartridge body 20 during theassembly of the microfluidic cartridge 1.

The first film 11 and the second film 12, thus applied on the substrate100 of the cartridge plate 10, form with it the fluidic network ofmicrochannels C1 to C15, C11 a to C16 a, C15 b, C16 b (see FIG. 12).

It will be seen hereinafter how the microchannels and the valves formedin the cartridge plate 10 are used to transport and transfer the fluidsrequired for the analysis of the sample.

As shown in FIGS. 8 and 11, the cartridge plate 10 also includes atleast two recessed cavities R1 a, R2 a separated from each other andformed in the substrate 100. These two recessed cavities R1 a, R2 a aremade in the first face 101 and extend from the latter toward the insideof the substrate 100 (see FIG. 8A).

The two recessed cavities R1 a, R2 a are tightly closed by the firstfilm 11 deposited on the first face 101 of the substrate 100, in such amanner to form two reaction chambers for nucleic acid amplification,called hereinafter amplification chambers and referenced AMP1 and AMP2(see FIG. 12).

The circulation of the fluids toward or out of these two amplificationchambers AMP1, AMP2 is controlled by the valves V11, V13 and by thevalves V12, V14, respectively.

The valves that control the circulation of the fluids between theamplification chambers (typically V13 and V14) are actuated by linearactuators that are independent of the cam-driven actuator.Preferentially the valves that control the circulation of the fluidstoward the amplification chambers (typically V11 and V12) are actuatedby linear actuators that are independent of the cam-driven actuator.

In the same way, as shown in FIGS. 8 and 11, the cartridge plate 10includes two other recessed cavities R1 b, R2 b and formed in thesubstrate 100.

These two recessed cavities R1 b, R2 b, substantially parallelepiped inshape, are made in the first face 101 and extend from the latter towardthe inside of the substrate 100. As shown in FIG. 8B, the two recessedcavities R1 b, R2 b have indented inclined flanks 105, 106,respectively.

These two recessed cavities R1 b, R2 b are tightly closed by a biochip110 (see FIG. 2) bonded on the first face 101 of the substrate 100, soas to form two reaction chambers for nucleic acid analysis, calledhereinafter hybridization chambers and referenced HYB1 and HYB2 (seeFIG. 12).

The circulation of the fluids toward or out of these two analysischambers HYB1, HYB2, is made through the through holes H7 a, H15 d (forthe hybridization chamber HYB1) and through the through holes H7 b, H16d (for the hybridization chamber HYB2), respectively.

In one embodiment, the cartridge may include, upstream from eachamplification chamber, a metering chamber, located between the centralHUB and each amplification chamber. For example said metering chambersare connected to the said amplification chambers through valves V11 andV12 (see FIG. 12) or VV8 and VV14 (see FIG. 16). Typically said meteringchambers are also connected to the central HUB, for example viamicrochannels C11 and C12. Alternatively, or additionally, said meteringchambers can also be directly connected, through a microchannel, to avalve of a hub-connected microchannel. Said metering chambers are usefulfor calibrating the proper fluid level to be injected to theamplification chambers.

The hybridizations chambers comprise an affinity biosensor for detectingthe presence of specific target molecules in the sample. The affinitybiosensors interact with the target molecule by ligation. The cartridgeaccording to the present invention is intended to allow the detection inparallel of the presence of several molecular hybridization markerswithin a biological sample. The capture of the amplification products,or amplicons, among a multiplicity of candidates on a surface is atechnique that is well known of the one skilled in the art, to perform amultiplexed detection. The favorite mode of detection is the biochip.The biochip systems are presently widely used for the detection and themeasurement of specific substances in complex samples. With such abiochip, the identity and quantity of a target DNA in a sample aremeasured by measuring the level of association of the target sequencewith probes specifically provided for said sequence. In the DNA biochiptechnologies, a set of probe nucleic acids, each having a definedsequence, is immobilized on a solid support or substrate in such a waythat each probe occupies a predetermined position.

According to the embodiment as exemplified in the present application,the biochip 110 essentially includes a solid substrate 111,approximately planar, for example a glass, silicon or plastic plate, onthe surface of which are immobilized probe molecules, whose sequence isspecific for target nucleic acids. As a matter of example the size of abiochip well suited for the cartridge of the invention is approximatelyof 24 mm×24 mm×0.1 mm.

The cartridge body 20 of the microfluidic cartridge 1 will now bedescribed with reference to FIGS. 1, 2 and 6.

Preferably, the cartridge body 20 is made separately from the cartridgeplate 10. In this case, the cartridge body 20 is made in threedimensions, advantageously by injection molding of a thermoplasticpolymer material such as polypropylene (PP).

In a variant, the cartridge body may be made out of cyclic olefincopolymers (COC) or cyclic olefin polymers (COP), in particular chosenin the group comprising polycarbonate, polyacrylamide, polyethylene,polymethyl-methacrylate (PMMA), polydimetyl-siloxane (PDMS), polyvinylchloride (PVC).

In some embodiments, the cartridge body is made in three dimensions forexample by stereolithography or by sintering.

According to another advantageous variant, the cartridge body and thecartridge plate may be fabricated together so as to form a single piece.In this case, said piece is made for example by injection molding usingthe same kind of materials used for the cartridge plate 10 and for thecartridge body 20.

When the microfluidic cartridge 1 is assembled (see FIG. 1), thiscartridge body 20 is in contact with the cartridge plate 10 on thesecond face 102 of the substrate 100, at the level of the first edge 22of the cartridge body 20.

As shown in FIG. 2, the cartridge body 20 includes a lateral wall 21extending perpendicular to the substrate 100, from the second face 102of this substrate 100 to a second edge 23 of the cartridge body 20.

The cartridge body 20 also includes a plurality of internal walls W0,W1, W2, W3, W4, W5, W6, W7, W8, W9, W10, which define a plurality offunctional volumes CT, T1, T2, T3, T4, T5, AMP, DET, T9, T10,respectively (see FIG. 2).

These different functional volumes CT, T1, T2, T3, T4, T5, AMP, DET, T9,T10, of the cartridge body 20 are containers intended to receive, duringthe use of the microfluidic cartridge 1 for the analysis of the sampleS, the sample S, which is treated or not, different reagent products, apurification column, as well as fluids or solids intended to thepreparation, the amplification and the analysis of the sample S.

The functions of these different functional volumes will be describedhereinafter.

Besides, as shown in FIG. 2, it is observed that the lateral wall 21 andthe six internal walls W0, W5, W6, W7, W8, W9, also define thefunctional volume WST that, as will be seen hereinafter, is a volume forthe wastes coming from the sample S and from the different reagentproducts.

When the microfluidic cartridge 1 is assembled (see FIG. 1), thecartridge body 20 being fixed to the cartridge plate 10 at the level ofthe second face 102, each functional volume T1, T2, T3, T4, T5, T9, T10,CT, WST, AMP, DET, is closed at the level of the first edge 22 of thecartridge body 20 by the first face 102 of the substrate 100, such that:

-   -   the functional volumes T1, T2, T3, T4, T5, T9, T10 comprise the        through holes H1, H2, H3, H4, H5, H9, H10, respectively;    -   the functional volume CT surrounds and comprises the whole of        the central through holes H0 c, H1 c, H2 c, H3 c, H4 c, H5 c, H9        c, H9 c, H10 c;    -   the functional volumes AMP et DET surround the two amplification        chambers AMP1, AMP2, and the two detection chambers HYB1, HYB2,        respectively;    -   the functional volume WST is in communication with the through        holes H7, H7 a, and H7 b.

Thus, it is understood (see in particular FIG. 12) that the functionalvolumes T1, T2, T3, T4, T5, T9, T10, are, each independently, in fluidiccommunication with the functional volume CT via the microchannels C1,C2, C3, C4, C5, C9, C10 controlled by the valves V1, V2, V3, V4, V5, V9,V10, and fluids can circulate, in one direction as in the other, betweenthese different functional volumes.

For that purpose, the functional volume CT, also called central tube,forms a hub body into which, or out of which, a syringe 60 (see FIGS. 1and 2) can slide.

More precisely, as shown in FIG. 4, the syringe 60 includes a plunger 62and a plunger seal 61, in which the plunger 62 is fixed. For example,the plunger 62 may be attached to the plunger seal 61 by inserting inforce the plunger 62 into the plunger seal 61 comprising deformableattaching means.

The plunger 62 also comprises, on the opposite side with respect to theplunger seal 61, a flat 63 making it possible to push or pull on thisplunger 62 to make the syringe 60 slide in the hub body CT.

The plunger seal 61 of the syringe 60 comprises two O-rings 61A, 61B andhas an outer diameter adjusted in such a manner that, once engaged inthe central tube CT, it can tightly slide in the central tube CT.

That way, the syringe 60 can pump or inject fluids in the differentfunctional volumes T1, T2, T3, T4, T5, T9, T10, that are connected tothe central tube CT through microchannels C1, C2, C3, C4, C5, C9, C10.

In a preferred embodiment, only the plunger seal 61 is part of thecartridge body 20 of the microfluidic cartridge 1. In this preferredembodiment, the plunger 62 of the syringe 60 is part of the dockingstation 1000. Therefore, the number of moving parts in the microfluidiccartridge 1 is reduced, like its cost of fabrication.

It will then be considered that the hub body CT and the syringe 60 arepart of a central distribution hub of fluids, hereafter called centralhub and referenced with the reference sign CH.

As can also been understood from FIG. 12, this central hub CH is alsocapable of pumping or injecting fluids:

-   -   from or to the waste container WST via the microchannel C7, and        thanks to the valve V7;    -   from or to the amplification chambers AMP1, AMP2, via the        microchannels C11, C11 a, C12, C12 a and thanks to the valves        V11, V12;    -   from or to the detection chambers HYB1, HYB2, via the        microchannels C6, C8, C15 a, C16 a, C15, C16, C15 b, C16 b,        thanks to the valves V6, V8.

In one embodiment of the microfluidic cartridge, the valves associatedwith the waste container or with the detection chambers are not locatedon hub-connected microchannels, and therefore are actuated by linearactuators that are independent of the cam-driven actuator.

As shown in Figures and 2, the cartridge cover 30 of the microfluidiccartridge 1 comes and inserts into the cartridge body 20, resting on itssecond edge 23 so as to close the different functional volumes T2, T4,T5, T9, T10, WST, AMP, DET.

As the cartridge plate 10 and the cartridge body 20, the cartridge cover30 is made advantageously by injection molding of a thermoplasticpolymer material such as polypropylene (PP).

In a variant, the cartridge cover may be made by injection molding of athermoplastic polymer material such as, for example, the cyclic olefincopolymers (COC) or the cyclic olefin polymers (COP), in particularchosen in the group comprising polycarbonate, polyacrylamide,polyethylene, polymethyl-methacrylate (PMMA), polydimetyl-siloxane(PDMS), polyvinyl chloride (PVC).

The cartridge cover 30 comprises venting holes 32 at the level of eachfunctional volume T2, T4, T5, T9, T10, so as to permit the suction andthe injection of fluids in these volumes of the central hub CH.

In an assembled configuration (FIG. 1), before the use of themicrofluidic cartridge 1 for the analysis of the sample S, the cartridgecover 30 comprises a protection film 31 tightly covering the whole ofthe venting holes 32, so as to protect the content of the functionalvolumes T2, T4, T5, T9, T10, during transportation or storage of themicrofluidic cartridge 1. This protection film 31 may be for examplemade of a plastic or metallic (e.g. aluminum) thin sheet.

In one embodiment, the microfluidic cartridge may further comprise asemi-permeable membrane between the cartridge body and the cartridgecover. This semi-permeable membrane comprises, on one side, ahydrophobic layer and, on the other side, an adhesive layer in order toseal the membrane to the second edge of the cartridge body.

The semi-permeable membrane acts as a GORETEX™ fabric, and is adapted tolet air pass through it while preventing liquids to leak out of thefunctional volumes. Therefore, this semi-permeable membrane allows theventing of the various functional volumes of the microfluidic cartridge.

As shown in FIGS. 1 and 2, the microfluidic cartridge 1 also includesherein two tubes 40, 50, which are assembled in the microfluidiccartridge 1 during the use thereof, by plunging into the tube T1 and thetube T3, respectively, of the cartridge body 20.

The first tube 40, that contains the sample S, is a sample tube thatcomprises (see FIG. 3) a body 42 of cylindrical shape, a cap 41 closingthe body 42 on one side of the tube, and a terminal opening 43 locatedon the other side of the tube. The cap 41 may contain a semi permeablemembrane allowing air flow while retaining liquids.

According to the embodiment as exemplified in the present application,the terminal opening 43 is here closed by a plastic bead 44 according toa technology similar to that of the disposable ink cartridges.

In particular, the container T1 intended to receive the sample tube 40comprises a suction head designed to push the plastic bead 44 so as toeject the plastic bead 44 from its blocking position, where it preventsthe flowing of the content of the sample tube 40.

In a variant, the sample may be injected directly into the container,either by using a tube without suction head or by pipetting the samplewith a micropipette or syringe into the dedicated container.

Besides, the sample tube 40 also comprises a filter 45 placed inside thebody 42 of the tube 40, so as to limit the quantity of large particles,coming from the sample S or from by-products of the sample S, enteringinto the microfluidic network.

The second tube 50 is a tube that comprises a mixture for theamplification reaction, referred to as amplification-mix tube, which ishas a shape similar to that of the first tube 40 with a body 51, a cover52, and a terminal part 53 also comprising a closing bead (not shown).

The above-described microfluidic cartridge 1 is intended to be insertedin a docking station 1000, a partial sectional view of which is shown inFIG. 13.

In the embodiment shown in FIG. 13, the docking station 1000 includes arotational-motion cam-driven actuator 1100.

More precisely, the cam-driven actuator 1100 includes a cam 1120 (seeFIG. 15), which is herein an annular cylindrical part 1121 around anaxis of revolution A1, which has a first surface 1121A and a secondsurface 1121B, substantially planar and parallel to each other, and acentral opening 1123.

Advantageously, the cam 1120 comprises on its first surface 1121 arectilinear cam recess 1124 extending along a radius of the cylindricalpart 1121. The profile of this cam recess 1124, considered along aperimeter of the annular part 1121, is herein curved and has, on thebottom of the cam recess 1124, a radius of curvature Rc.

The cam-driven actuator 1100 also comprises a planar guiding plate 1110(see FIGS. 13 and 14), herein perforated with ten cylindrical holes 1111arranged circularly and passing perpendicularly through the guidingplate 1110. These guiding holes 1111 are intended to guide ten actuatingballs 1102 of the cam-driven actuator 1100 (see FIG. 13, where only oneactuating ball 1102 is shown), such actuating balls having a balldiameter adjusted so that they can slide through said guiding holes 1111without rubbing excessively on the walls of these latter.

As shown in FIG. 13, the guiding plate 1110 of the rotational-motioncam-driven actuator 1100 is located above the cam 1120 so that theactuating balls 1102 rest on the first surface 1121A of the cam 1120.

Besides, the radius of the cylindrical holes 1111, and thus the diameterof the actuating balls, is adjusted with respect to the thickness of theguiding plate 1110 so that:

-   -   in the case where an actuating ball 1102 rests on a planar part        of the first surface 1121A of the cam 1120 (the case of FIG.        13), the actuating ball 1102, maintained in place by its        corresponding cylindrical hole 1111, projects upward from the        guiding plate 1110;    -   in the case where an actuating ball 1102 rests on the cam recess        1124 of the annular part 1121 of the cam 1120, the actuating        ball 1102, guided by its corresponding cylindrical hole 1111,        projects downward from the guiding plate 1110.

Therefore, upon a rotational motion of the cam 1120 around the axis ofrevolution A1, the actuating ball 1102 performs a translation motionparallel to said axis of revolution A1, the actuating ball 1102 beingguided thanks to the corresponding cylindrical hole 1111 between anengaged position where the actuating ball 1102 projects from thecylindrical hole 1111, while moving far from the cam 1120, and adisengaged position where the actuating ball 1120 move closer to the cam1120.

In the preferred embodiment shown in FIG. 15, where the cam 1120comprises only one cam recess 1124, at most one actuating ball 1102 canbe in a disengaged position while the other actuating balls 1102 are inan engaged position.

As shown in FIG. 13, the cam actuator 1100 comprises a series of tenplungers 1101 each located above an actuating ball 1102 and anotherguiding plate 1130, similar to the first guiding plate 1110, which isalso perforated so as to guide the plungers 1101 in their translationmotion.

In the cam-driven actuator 1100, the cylindrical holes 1111, theactuating balls 1102 and the plungers 1101 are arranged circularly.

In the microfluidic cartridge 1 according to the invention, the tenvalves V1 to V10 are arranged so as to be mechanically actuated togetherby the external cam-driven actuator 1100.

More precisely, the valve V1 to V10 of the microchannels C1 to 010connected to the central hub CH are arranged circularly so that there isa plunger 1101 opposite each of the valves V1 to V10.

So arranged, it is understood that:

-   -   when an actuating ball 1102 is in an engaged position (the case        of FIG. 13), it places the corresponding plunger 1101 also in an        engaged position where it exerts a pressure on the first film 11        of the cartridge plate 10, opposite the valve seat R1 of the        valve V1, so as to deform the first film 11 and to seal the        through hole H1, thus closing the valve V1, and    -   on the contrary, when an actuating ball 1102 is in a disengaged        position, the plunger 1101 also goes to a disengaged position        where it does not exert any more pressure to the first film 11        of the cartridge plate 10, so that the first film 11 is at rest        opposite the valve seat R1 of the valve V1, so that the valve V1        is then open.

Therefore, as seen above, it is understood that the cam-driven actuator1100 allows the opening of at most one valve V1 to V10 at the same timewhen the microfluidic cartridge 1 is inserted in the docking station1000 and when the microfluidic station 1 is actuated by the cam-drivenactuator 1100.

Although it is not shown, the docking station 1000 in the embodimentshown in FIG. 13 also includes sliding means to make the syringe 60 ofthe central hub CH slide into and out of the hub body CT.

These sliding means may comprise, for example, the plunger 62 of thesyringe 60 and a fork-shape lever that catches the plunger 62 of thesyringe 60, below the flat 63, so as to lower down or lift up theplunger 62.

Besides, as already known, the docking station 1000 also comprises:

-   -   optical excitation means for exciting the biochip 110 in contact        with the two hybridization chambers HYB1, HYB2, and    -   optical detection means for detecting an optical signal emitted        from the hybridization chambers HYB1, HYB2 and that is        representative of the at least one nucleic acid searched in the        sample S analyzed by the microfluidic cartridge 1.

In this embodiment wherein the detection biochip 110 is used, thedetection and quantification of the interaction between the targetmolecules and the probe is made to a device for optical detection: lightradiation of a first wavelength excites chromophores linked to thetarget molecules. The light emitted by the chromophore at a secondwavelength in response to their excitation light is then collected by acollection device.

It is also particularly advantageous that the present microfluidiccartridge 1, and thus the reading of the biochip 110, be suitable for asystem for collecting the light emitted by the chromophore in responseto light excitation type contact imaging.

It can be considered that the microfluidic cartridge 1 is intended to beplaced in an apparatus for reading optical contact imaging. Such contactimaging devices have been notably described in WO 2004042376, WO2004068124, WO 2007045755, WO 2010007233 and WO 2012089987.

Advantageously, the substrate 100 is transparent.

In the case of detection of target nucleic acids by means of a biochip110 fluorescence, it may be advantageous that the substrate of thebiochip 110 may comprise fluorescent substances immobilized on itssurface which absorbs light at a first excitation wavelength and emitlight at a second wavelength transmission, comprises means forincreasing the efficiency of the amount of light emission based on theamount of excitation light.

A method intended to be implemented by an operator in order to analyzethe sample S contained in the sample tube 40, said tube being insertedin the functional volume T1 of the microfluidic cartridge 1 (see FIG.2), will now be described.

The sequence of operations performed by the diagnostics machine maycomprise the following steps:

-   -   DNA extraction & purification from the lysed sample    -   DNA amplification using any amplification method including but        not limited to Polymerase Chain Reaction (PCR), Reverse        transcriptase PCR and isothermal amplification;    -   Hybridization on a microarray using highly-specific probes (such        as the HairLoop™ probes) or standard linear probes to        discriminate markers up to SNP discrimination level.    -   Detection of hybridization by fluorescent labeling using a        fluorescence integrated reader preferentially allowing        contact-imaging devices integrated to the docking station such        as described in WO 2004042376, WO 2004068124, WO 2007045755,        WO2010007233 and WO 2012089987.

In one embodiment, a pre-lysis step is performed prior injection of thesample in the cartridge.

Lysis buffer and/or reagents can be added to the sample prior injectionin the cartridge and/or stored in one functional volume of thecartridge, as lyophilized pellet.

The sample may be a solution or in suspension, in particular the samplemay be a bodily fluid, such as feces, whole blood, plasma, serum, urine,sputum, saliva, seminal fluid, mucus and cerebrospinal fluid. The samplemay also be a solid made soluble or suspended in a liquid.

By nucleic acid it is intented according to the present invention, anysynthetic or naturally occurring nucleic acid in any configuration(single-stranded or double-stranded DNA).

It is noted that in some embodiments of the invention, the targetnucleic acid may be in the form of RNA in the sample, typically whenviral nucleic acid are searched in the sample to analyze. In suchembodiment the nucleic acid may be subjected to RT PCR.

The main steps of this analysis method are performed in the functionalvolumes of the microfluidic cartridge 1 which comprises a plurality offunctional areas comprising at least:

-   -   a sample preparation area comprising the different functional        volumes designed to extract a specific nucleic acid from the        sample S to analyze;    -   a nucleic acid amplification area comprising the functional        volumes adapted to perform the amplification of the nucleic acid        contained in the sample S. According to various embodiments, the        nucleic acid amplification area comprises one or more        amplification chambers;    -   a nucleic acid analysis area, typically comprising the        functional volume T9 and T10. According to various embodiments,        the nucleic acid amplification area comprises one or more        detection chamber; and    -   a waste area comprising the functional volume WST designed for        the waste.

In the preferred embodiment described above, the different functionalareas are such that:

-   -   the sample preparation area comprises the sample tube 40, the        amplification-mix tube 50, and the three functional volumes T2,        T4, T5 comprising respectively a purification column, a first        DNA wash buffer and a second DNA wash buffer;    -   the nucleic acid amplification area comprises the two        amplification chambers AMP1, AMP2;    -   the nucleic acid detection area comprises the two functional        volumes T9 and T10 comprising, respectively, a buffer for        hybridization and a hybridization wash buffer.

Therefore, according to the embodiment of the microfluidic cartridge 1described above in reference to FIGS. 1 and 2, the sample preparationarea and the waste area are directly fluidly connected to the centralhub CH of fluid distribution by one or more hub-connected microchannelsand one or more cam-driven actuated valves. The nucleic acid detectionarea may optionally be directly fluidly connected to the central hub CHby one or more hub-connected microchannels (C15 and C16).

Still in the embodiment of the microfluidic cartridge 1 described inreference to FIGS. 1 and 2, the amplification area is not directlyconnected to the central hub CH and fluidic connection toward this areaand notably to each amplification chamber is controlled by at least onevalve actuated by a linear actuator.

As explained above, the central hub CH by means of the syringe 60engaged in the central tub CT is able to transfer fluids from a firstfunctional area to a second functional area of the plurality offunctional areas by passing through it.

In the way the different functional areas are arranged in themicrofluidic cartridge 1, said second functional area can be eitheridentical or different from said first functional area.

Moreover, one will understand with the following description of theanalysis method how the plurality of functional areas cooperates witheach other in order to analyze the sample S.

Step a)

In a first step (step a), the operator provides the biological sample Sinto at least one functional volume of the sample preparation area of amicrofluidic cartridge 1, namely here in the sample tube 40 which isinserted into the microfluidic cartridge 1.

At start-up, the sample tube 40 may already contains a lysis buffer.Disruption of most cells may be done by chaotropic salts, detergents oralkaline denaturation. The lysis of the sample S is typically performedthrough a Lysis and Proteinase K Buffer already present in the sampletube 40 when injecting the sample S into this tube 40.

Once the microfluidic cartridge 1 is inserted in the docking station1000, the sample S is incubated during a few minutes to completely breakdown cellular membranes by the chemical lysis. The Proteinase K bufferfinishes the digestion of protein cellular components.

In another embodiment, lysis buffer and reagents (such as protein Kbuffer) may also be stored in a functional volume of the cartridge aslyophilized pellet.

In another embodiment, where the sample comprises hard-to-treat matrixor microorganisms, the following steps might be necessary beforeinsertion of the sample preparation tube into the microfluidiccartridge:

-   -   lysis of the sample using for example bashing beads together        with a specific cell disruption buffer;    -   vortex and heat during a few minutes, for example 5 minutes, at        a temperature up to 70° C.;    -   addition of Binding Buffer and potentially of a reagent allowing        amplification inhibitor absorption, such as InhibitEX Matrix, to        the sample preparation tube.

Step b)

The sample S is put into contact with a reagent typically present in thepurification column T2.

For this, the cam-driven actuator 1100 is rotated by the docking station1000 and put in a position so as to actuate consecutively the valve V1and the valve V2 in the following way:

-   -   valve V1 open and valve V2 closed: the plunger 62 of the syringe        60 is slid out of the central tube CT by the fork-shape lever of        the docking station 1000 in order to pump the lysed sample S        from the sample tube 40 to the central tube CT;    -   valve V1 closed and valve V2 open: the plunger 62 of the syringe        60 is slid in the central tube CT by the fork-shape lever of the        docking station 1000 in order to inject the lysed sample S from        the central tube CT into the purification column T2.

The purification column T2 may contain a silica-like membrane for DNAbinding.

According to various embodiments, the purification column may forexample contain a gel, beads, or a paper filter for DNA binding andconcentration. As a matter of illustration, agarose gel, silica beadsand filter paper, such as cellulose, base purification may also be usedaccording to the invention.

Once the binding is completed, the sample S is re-aspirated from thepurification column (valve V2 open), and disposed to the waste areathrough the central hub CH with valve V7 open, while the DNA is retainedby the purification column T2.

Step c)

In this step, the product resulting of step b) is recovered by washingit in order to remove inhibitors and purify the DNA.

In the embodiment as exemplified in the present application, the bindingmembrane of the purification column T2 is washed successively by one ormore DNA wash buffers, typically two, as contained in the functionalvolumes T4, T5.

To this end, firstly, the valve V4 is open (all other valves beingclosed) by the cam-driven actuator 1100 and the first DNA wash buffercontained in the functional volume T4 is pumped out by the central hubCH and then the valve V2 is open (valve V4 being therefore automaticallyclosed) and the central hub CH injects the first DNA wash buffer in thepurification column T2.

Secondly, the same operation is repeated with the second DNA wash buffercontained in the functional volume T5 (valve V2 closed/valve V5 open andthen valve V2 open/valve V5 closed).

Thirdly, the DNA bound to the binding membrane is eluted with an elutionbuffer. The amplification-mix solution contained in theamplification-mix tube T3 can be used as an elution buffer. For that,valve V2 is closed, valve V3 is opened thanks to the rotation of thecam-driven actuator 1100 of the docking station 1000, and the centralhub CH sucks out the amplification-mix solution into the central tubeCT; then valve V3 is closed, valve V2 is opened, and the syringe 60 isslid into the central tube CT so that the central hub CH injects thePCR-mix solution into the purification column 20.

At the end of step c), one obtains an isolated DNA sample.

Step d)

After elution, the isolated DNA sample amplification-mix is transferredinto the two amplification chambers AMP1, AMP2 for amplification.

For that, the isolated DNA sample is pumped from the purification columnT2 (valve V2 still open) to the central tube CT.

Then, all valves V1, V2, V3, V4, V5, V6, V7, V8, V9, V10 are closed byactuation of the cam-driven actuator 1100.

The valve V11 and V12 of the microfluidic cartridge 1, which areindependently actuated by two standard linear actuators, are opened,allowing of the isolated DNA sample to go through the micro-channelsC11, C11 a, C12, C12 a to the two amplification chambers AMP1, AMP2.

In some embodiments, the isolated DNA sample amplification-mix istransferred to a metering chamber prior to transfer to eachamplification chamber (AMP1 and AMP2), in order to calibrate the propervolume to be injected in the said amplification chambers.

Step e)

During this step, after the valves V11, V12 of the nucleic acidamplification area have been closed, the isolated DNA sample is put intocontact with a reagent for amplification.

Step f)

The DNA amplification in the amplification chambers AMP1, AMP2 isperformed by standard amplification protocols of the prior art(typically any amplification method including but not limited toPolymerase Chain Reaction (PCR), Reverse transcriptase PCR andisothermal amplification) achieving a very good sensitivity andspecificity up to 20 markers.

In each amplification chamber AMP1, AMP2, a separate set of primers havetypically been immobilized during the manufacturing process. Theseprimers are re-suspended when the amplification chambers AMP1, AMP2 arefilled by a ready-to-use solution typically containing polymerase,nucleotides and reaction buffers at optimal concentrations for efficientamplification of DNA templates.

At the end of this step, one obtains an amplified DNA sample.

Step q)

In this step, the hybridization buffer contained in the functionalvolume V10 is transferred through the central hub CH to the twohybridization chambers (that can also be named detection chambers) HYB1,HYB2.

To this end, valve V10 is opened (all other valves V1 to V9 beingclosed) by the rotational-motion cam-driven actuator 1100 of the dockingstation 1000 and transferred to the central tube CT.

Then, valves V6 and V8 may successively be opened in order to proceed tothe pre-filling of the hybridization chambers HYB1, HYB2 with thehybridization buffer if necessary.

Amplification chamber valves are opened and amplification solution ispushed to the hybridization chambers HYB1, HYB2.

The amplified DNA sample is then put into contact with hybridizationbuffer upon opening of the valves V13, V14 (which are typicallyindependently actuated) into the hybridization chamber HYB1, HYB2through the area-connecting micro-channels C13, C15B, C14, C16 bconnecting directly the two functional areas, namely the nucleic acidamplification area and the nucleic acid hybridization area.

Then, the valves V13, V14 are finally closed.

Step h)

In this step, the sample is placed into contact with the affinity sensor(e.g. the biochip) in such a way that the complementary sequences can becombined with an immobilized probe, for example by hybridization,association or linking to the probe. After the elimination of thenon-associated material, the associated sequences are ready fordetection and measurement.

Typically, in this step the amplified DNA sample is hybridized, duringseveral minutes, e.g. about 30 minutes, in the hybridization chambersHYB1, HYB2. Recovering of the hybridized DNA is made by transferring thehybridization wash buffer contained in the functional volume T9 throughthe central hub CH to the hybridization chambers HYB1, HYB2. Ahybridized DNA sample is therefore obtained.

In a variant of the analysis method, a DNA melting procedure at the endof hybridization may be added and would allow an increase in detectionspecificity.

Step i)

In this step, a microarray image is obtained and analyzed. It is notedthat in accordance with the paragraph below, the hybridization chamberscan therefore also be named detection chambers.

The detection of the interaction between the target nucleic acids andthe probes are performed by an optical detection device. The localizedhybridization is detected by the emission of a chromogenic signal.Herein, “chromogenic signal” is to be understood as any light signalemitted directly, or indirectly, after excitation by a suitable lightsource or after chemical or enzymatic transformation. Hence, areincluded in the category of the chromogenic signals, the colorimetric,photoluminescent, fluorescent, chemoluminescent, bioluminescent signals,or the like. Such signals are either directly emitted by the moleculesof interest, or emitted by detectable elements (tags), which are addedand/or grafted thereto.

A fluorescence reader can therefore allow obtaining a fluorescent imageof the biochip surface. For that purpose, the biochip is illuminatedwith a light source at the wavelength of excitation of the fluorophoremarking the target molecules, and an adapted optical system forms animage of the fluorescence of the biochip at the wavelength of emissionof the fluorophores. The light intensity of each point of this image isrelated to the quantity of fluorophores present at the correspondingpoint of the biochip, which is itself proportional to the number oftarget molecules that have been selectively attached at this placeduring the hybridization phase, which makes it possible to collectinformation (often quantitative) about the nucleic acid content of thesample. Detection of the signal is preferentially achieved by contactimaging forming a compact readout optical system as described forexample in documents U.S. Pat. No. 7,306,766, FR2932885, US20050201899,PCT/FR2011/053208.

An automated analysis of the microarray image and a diagnostic report isthen generated about the analysis of the biological sample.

Many different configurations are possible within the scope of thisinvention, including variations on part geometries, materials, methodsof assemblies and configurations of parts relative to each other. Thedescription above is meant to illustrate and represent one possibleembodiment of the invention, and should not be construed to limit thepossible scope of variations.

For example, in the embodiment illustrated on FIG. 16, the microfluidiccartridge comprises a cartridge plate 2010.

The cartridge plate 2010 of the microfluidic cartridge comprises here aplurality of twelve valves VV1, VV2, VV3, VV4, VV4, VV5, VV6, VV7, VV8,VV9, VV10, VV1, VV12, each valve VV1, VV2, VV3, VV4, VV4, VV5, VV6, VV7,VV8, VV9, VV10, VV1, VV12 being located on a microchannel CC1, CC2, CC3,CC4, CC5, CC6, CC7, CC8, CC9, CC10, CC11, CC12 connected to the centraldistribution hub CH of fluids.

In this variant, all those twelve valves VV1, VV2, VV3, VV4, VV4, VV5,VV6, VV7, VV8, VV9, VV10, VV1, VV12 are arranged on a circle CR (seeFIG. 16) on the cartridge plate 2010 in order to be actuatedmechanically by an external cam-driven actuator.

The cartridge plate 2010 also comprises two couples of valves VV13,VV14, VV15, VV16 that may be actuated by independent linear actuators inorder to transfer fluids, for example, from the sample preparation areato the nucleic acid amplification area AMP1, AMP2 and the nucleic acidanalysis area HYB1, HYB2.

Typically in this embodiment, the cartridge comprises metering chambersthat are located between the central HUB and each amplification chamber.Said metering chambers can be connected to the said amplificationchambers through valves VV8 and VV14. Typically said metering chambersare also connected to the central HUB. Additionally, said meteringchambers can also be directly connected, through a microchannel, to avalve of a hub-connected microchannel (for example valves VV9 and VV7).

The person skilled in the art would adapt other elements of themicrofluidic cartridge, e.g. the cartridge body and the cartridge cover,in order to adapt the cartridge plate 2010 to the different functionalvolumes of the microfluidic cartridge.

1-25. (canceled)
 26. A microfluidic cartridge (1) for detecting at leastone nucleic acid of a sample (S), said microfluidic cartridge (1)comprising: a plurality of functional volumes split into functionalareas such as, at least, a sample preparation area (T1, T2, T3, T4, T5),a nucleic acid amplification area (AMP1, AMP2), a nucleic acid analysisarea (T9, T10, HYB1, HYB2), a waste area (WST), and a fluidic network ofmicrochannels (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13,C14, C15, C16, Clla, C12 a, C15 a, C15 b, C16 a, C16 b), wherein: atleast three functional areas are fluidly connected to one centraldistribution hub (CH) of fluids by one or more hub-connectedmicrochannels (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12), eachof said hub-connected microchannels having a hub end and an area end,said central distribution hub (CH) being capable of pumping andinjecting fluids from a first functional area to a second functionalarea of said at least three functional areas by passing through saidcentral distribution hub (CH), said second functional area beingidentical or different from said first functional area, and at leastthree valves (V1, V2, V3, V4, V5, V6, V7, V8, V9, V10) each located on ahub-connected microchannels are arranged in said microfluidic cartridge(1) so that said at least three valves (V1, V2, V3, V4, V5, V6, V7, V8,V9, V10) are adapted to be actuated mechanically by a single externalcam-driven actuator (1100).
 27. The microfluidic cartridge (1) accordingto claim 26, wherein the at least three valves (V1, V2, V3, V4, V5, V6,V7, V8, V9, V10) are circularly arranged in the microfluidic cartridge(1).
 28. The microfluidic cartridge according to claim 26, wherein theat least three valves are linearly arranged in the microfluidiccartridge.
 29. The microfluidic cartridge (1) according to claim 26,wherein each hub-connected microchannel (C1, C2, C3, C4, C5, C6, C7, C8,C9, C10, C11, C12) comprises one valve (V1, V2, V3, V4, V5, V6, V7, V8,V9, V10, V11, V12) located at the area end of said hub-connectedmicrochannel (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12). 30.The microfluidic cartridge (1) according to claim 26, wherein twofunctional areas (AMP1, AMP2, T9, T10, HYB1, HYB2, WST) of the pluralityof functional areas are directly fluidly connected to each other by oneor more area-connecting microchannels (C13, C14), each of saidarea-connecting microchannels (C13, C14) having a valve (V13, V14). 31.The microfluidic cartridge (1) according to claim 26, comprising: acartridge plate (10) comprising: a substrate (100) having a first face(101) and a second face (102), a plurality of grooves (G1, G2, G3, G4,G5, G6, G7, G8, G9, G10, G11, G11 a, G12, G12 a, G13 a, G14 a, G15, G15a, G15 b, G16, G16 a, G16 b) flush with the first face (101) or thesecond face (102) and a plurality of through holes (H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H0 c, H1 c, H2 c, H3 c, H4 c,H5 c, H9 c, H10 c, H7 a, H7 b, H11 a, H12 a, H15A, H15 b, H15 c, H15 d,H16 a, H16 b, H16 c, H16 d) connecting said first face (101) and saidsecond face (102), and a first film (11) bonded on first face (101) ofsaid substrate (100) of the cartridge plate (10), said grooves (G1, G2,G3, G4, G5, G6, G7, G8, G9, G10, G11, G12) flush with the first face(101) being sealed by said first film (11) to form the hub-connectedmicrochannels (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12), saidfirst film (11) being a deformable membrane adapted to be deformed by anexternal actuator (1100), a cartridge body (20) in contact with thecartridge plate (10) on the second face (102) of said substrate (100),said cartridge body (20) comprising: a lateral wall (21) which extendsfrom the second face (102) of said substrate (100), and a plurality ofinternal walls (W0, W1, W2, W3, W4, W5, W6, W7, W8, W9, W10) whichdefines a plurality of functional volumes (CT, T1, T2, T3, T4, T5, AMP,DET, WST) of said cartridge body (20), and a cartridge cover (30)adapted to close the different functional volumes (CT, T1, T2, T3, T4,T5, AMP, DET, WST).
 32. The microfluidic cartridge (1) according toclaim 31, wherein the microfluidic cartridge (1) comprises asemipermeable membrane between the cartridge body (20) and the cartridgecover (30) adapted to let air pass through it while preventing liquidsto leak out of the functional volumes (CT, T1, T2, T3, T4, T5, AMP, DET,WST).
 33. The microfluidic cartridge (1) according to claim 31, whereinthe cartridge plate (10) comprises a second film (12) bonded on secondface (102) of the substrate (100) of the cartridge plate (10), theplurality of grooves (Gila, G12 a, G13 a, G14 a, G15 a, G15 b, G16 a,G16 b) flush with the second face (102) being sealed by said second film(12) to form the area-connected microchannels (C13, C14).
 34. Themicrofluidic cartridge (1) according to claim 31, wherein the cartridgeplate (10) comprises at least one recessed cavity (R1 a, R2 a, Rib, R2b) formed in the substrate (100) and extending from the first face (101)and a microarray slide (110) bonded on the first face (101) of thesubstrate (100) closes said at least one recessed cavity (Rib, R2 b) toform at least one detection chamber (HYB1, HYB2) for nucleic acidanalysis.
 35. The microfluidic cartridge (1) according to claim 31,wherein the functional volumes (CT, T1, T2, T3, T4, T5) of the cartridgebody (20) are containers adapted to receive tubes, sample (T1), reagentproducts (T3, T4, T5), or a purification column (T2).
 36. Themicrofluidic cartridge (1) according to claim 31, wherein the centraldistribution hub (CH) of fluid distribution comprises a hub body (CT)and a plunger seal (61) adapted to slide in and out of the hub body (CT)to pump from or inject fluids in the functional areas (T1, T2, T3, T4,T5, AMP1, AMP2, T9, T10, HYB1, HYB2, WST) of said microfluidic cartridge(1) through the hub-connected microchannels (C1, C2, C3, C4, C5, C6, C7,C8, C9, C10, C11, C12).
 37. The microfluidic cartridge (1) according toclaim 36, wherein the central distribution hub (CH) of fluiddistribution comprises a syringe (60) having a plunger (62) to which theplunger seal (61) is attached.
 38. A docking station (1000) adapted toreceive a microfluidic cartridge (1) according to claim 26, comprising acam-driven actuator (1100) adapted to actuate mechanically the at leastthree valves (V1, V2, V3, V4, V5, V6, V7, V8, V9, V10) of hub-connectedmicrochannels (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12).
 39. Adocking station (1000) adapted to receive a microfluidic cartridge (1)according to claim 26, comprising: means for optical excitation of themicroarray slide (110) of said microfluidic cartridge (1), and means foroptical detection of an optical signal that is representative of saidnucleic acid in the sample analyzed by the microfluidic cartridge (1),and. means for actuation adapted to actuate the other valves (V11, V12,V13, V14) of said microfluidic cartridge (1).
 40. The docking station(1000) according to claim 38, wherein the cam driven actuator (1100) isa rotational motion actuator.
 41. The docking station (1000) accordingto claim 40, comprising sliding means adapted to slide a syringe (60) ofa central distribution hub (CH) in and out of an hub body (CT) to pumpfrom or inject fluids in functional areas of said microfluidic cartridge(1) through the hub-connected microchannels (C1, C2, C3, C4, C5, C6, C7,C8, C9, C10, C11, C12).